OCCURRENCE AND METABOLISM OF 20ALPHA-HYDROXYCHOLESTEROL IN BOVINE ADRENAL PREPARATIONS

Using tritiated 20α-hydroxycholesterol and its sulfate as internal standards, 5 kg of bovine adrenal glands was processed to determine their content of these compounds. Double-isotope dilution techniques indicated the presence of 37 μg of unesterified 20α-hydroxycholesterol/kg of tissue. The compound was also found in higher concentration (70 μg/kg) in the fatty acid esterified fraction; on the other hand, the sulfate ester fraction was found to be devoid of this sterol. Quantification and identity were achieved using [14C]acetic anhydride and crystallization with carrier material both as the acetate and as the 3β-acetoxy 5,6-oxide. Incubation of the tritium-labeled steroid with sonicated preparations of adrenal mitochondria, resulted in rapid and complete conversion of the substrate into radioactive pregnenolone, 17-hydroxypregnenolone and to more polar products. When incubation experiments were carried out with the tritiated 20αhydroxycholesterol together with [14C]cholesterol, the recovered, unchanged hydroxysterol was devoid of 14C even though the isolated pregnenolone contained both isotopes. Although the sulfurylated hydroxysterol could not be found in adrenal tissue, the doubly labeled conjugate, when incubated with adrenal mitochondrial preparations that had been disrupted by ultrasound, was found to be converted, in high yield, into pregnenolone sulfate without any alteration of the 3H/35S ratio. © 1969, American Chemical Society. All rights reserved.